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  • MicroRNAs in the moss Physcomitrella patens

    Tzahi Arazi

    Received: 13 December 2010 / Accepted: 25 February 2011

    Springer Science+Business Media B.V. 2011

    Abstract Having diverged from the lineage that lead to

    flowering plants shortly after plants have established on

    land, mosses, which share fundamental processes with

    flowering plants but underwent little morphological chan-

    ges by comparison with the fossil records, can be consid-

    ered as an evolutionary informative place. Hence, they are

    especially useful for the study of developmental evolution

    and adaption to life on land. The transition to land exposed

    early plants to harsh physical conditions that resulted in

    key physiological and developmental changes. MicroRNAs

    (miRNAs) are an important class of small RNAs (sRNAs)

    that act as master regulators of development and stress in

    flowering plants. In recent years several groups have been

    engaged in the cloning of sRNAs from the model moss

    Physcomitrella patens. These studies have revealed a

    wealth of miRNAs, including novel and conserved ones,

    creating a unique opportunity to broaden our understanding

    of miRNA functions in land plants and their contribution to

    the latters evolution. Here we review the current knowl-

    edge of moss miRNAs and suggest approaches for their

    functional analysis in P. patens.

    Keywords AGO1 DCL1 Development Evolution Gametophyte MicroRNA Moss Physcomitrella patens Stress

    Introduction

    MicroRNAs, are genome-encoded noncoding RNAs of*21nucleotides (nt) in length that act as repressors of target genes

    in animals and plants (Bartel 2004). A mature miRNA and its

    passenger strand (miRNA*) are derived from opposing arms

    of a hairpin precursor (pre-miRNA) found in a longer pri-

    mary transcript (pri-miRNA) that is transcribed from MIR

    genes by RNA polymerase II (Chen 2008). In Arabidopsis

    thaliana, release of a pre-miRNA from the pri-miRNA and

    its subsequent processing into a mature miRNA/miRNA*

    duplex occurs via at least two cleavage steps, which are

    catalyzed by the RNaseIII-type enzyme DICER-LIKE 1

    (DCL1) (Kurihara et al. 2006; Kurihara and Watanabe 2004;

    Park et al. 2002) assisted by HYPONASTIC LEAVES1 and

    SERRATE (Dong et al. 2008; Kurihara et al. 2006). Fol-

    lowing its release, the miRNA/miRNA* duplex is methyl-

    ated by HUA ENHANCER 1 (Yu et al. 2005). Then, its two

    strands are separated and the single-stranded mature miRNA

    is specifically sorted into an ARGONAUTE (AGO) protein

    that form the core of an effector complex called RNA-

    induced silencing complex (RISC) (Mi et al. 2008). AGO1 is

    considered the major miRNA-RISC. It performs slicer

    activity that cleaves miRNA targets (Baumberger and

    Baulcombe 2005; Qi et al. 2005) and preferentially associ-

    ates with 21-nt-long sRNAs that have a 50 terminal uridine(Mi et al. 2008; Montgomery et al. 2008), features which are

    characteristic of most plant miRNAs. Loaded miRNAs guide

    RISC to nearly complementary target sequences resulting in

    their cleavage (Llave et al. 2002; Tang et al. 2003) or

    translation repression (Brodersen et al. 2008; Chen 2004).

    Accumulating evidence on miRNA functions in A. thaliana

    and other model flowering plants suggests that they

    play critical roles in vegetative and reproductive develop-

    ment (Chen 2009), nutrient homeostasis, response to

    T. Arazi (&)Institute of Plant Sciences, Agricultural Research Organization,

    Volcani Center, PO Box 6, Bet Dagan 50250, Israel

    e-mail: tarazi@agri.gov.il

    123

    Plant Mol Biol

    DOI 10.1007/s11103-011-9761-5

  • environmental stresses (Sunkar 2010), and autoregulation of

    the miRNA pathway itself (Vaucheret et al. 2006; Xie et al.

    2003).

    Bryophytes are believed to have shared a common

    ancestor with flowering plants *400 million years ago(MYA) (Kenrick and Crane 1997). Physcomitrella patens

    (Bryopsida) is a monoecious moss that has emerged as a

    useful model plant mainly because it is easily transformed

    and performs efficient homologous recombination, which

    allows the study of gene function by targeting gene dis-

    ruptions or replacements (Cove 2005; Schaefer 2001).

    Moreover, its complete genome sequence (*511 Mb) wasrecently published (Rensing et al. 2008) and powerful

    molecular tools are available (Frank et al. 2005a). Unlike

    vascular plants (ferns and seed plants), the life cycle of

    P. patens like all mosses is dominated by a haploid

    gametophyte phase (reviewed in Reski 1998) making the

    analysis of engineered loss-of-function P. patens mutants

    straightforward. Furthermore, the P. patens gametophyte

    has simple tissue morphology with only a few cell types,

    which facilitate the characterization of abnormal mutant

    phenotypes and the study of plant development (Prigge and

    Bezanilla 2010).

    The development of the dominant P. patens gameto-

    phyte, which is larger and more complex than the sporo-

    phyte, can be divided into two distinct stages: the

    protonema (juvenile gametophyte) and the gametophore

    (adult gametophyte) (Fig. 1). The protonema, generated by

    spore germination, is composed of filaments of cells that

    extend by successive divisions of their tip cell (Menand

    et al. 2007). Young protonema filaments have assimilatory

    functions and consist of chloronema cells that are densely

    packed with large chloroplasts and have perpendicular cell

    walls (Fig. 1a). These filaments extend until, in response to

    increases in light (Cove and Ashton 1988) and auxin (Johri

    and Desai 1973) their tip cells differentiate into caulonema

    cells, which are longer, divide more often, contain fewer

    smaller chloroplasts and have oblique cell walls (Fig. 1b).

    Soon after the division of a caulonema tip cell, an initial

    cell is formed in the second subapical cell. In the presence

    of cytokinin this initial cell, instead of producing a lateral

    filament, will divide and produce a bud, marking the

    transition from juvenile to adult gametophyte stage

    (Schumaker and Dietrich 1998) (Fig. 1c). By meristematic

    growth, this bud forms a leafy gametophore on which

    female and male gametangia will later develop (Fig. 1d).

    Once fertilized, the zygote will develop into a tiny diploid

    sporophyte that produces haploid spores (Fig. 1e).

    Cloning efforts have identified numerous miRNAs from

    various P. patens gametophyte tissues, many of which

    target regulatory genes. Furthermore, certain miRNA

    families and their corresponding targets have been found to

    be conserved with flowering plants, suggesting a common

    origin of miRNA-regulated pathways in land plants (Axtell

    and Bowman 2008). In this article, we review what is

    known about P. patens miRNAs and discuss how miRNA

    functions and regulatory roles may be elucidated in this

    unique model plant.

    miRNA discovery in P. patens

    The first experimental evidence of miRNA-guided target

    cleavage in a non-flowering plant was provided by Floyd

    Fig. 1 A cartoon of major stages in P. patens gametophyte devel-opment. The juvenile gametophyte stage is initiated by the germina-

    tion of a haploid spore (circle) to form a chloronema filament (a).Under certain conditions, a tip chloronema cell will differentiate into

    a caulonema cell (marked by an arrowhead, b). c In the presence ofcytokinin a juvenile gametophyte undergoes transition indicated by

    the formation of a bud (marked by an arrowhead). This bud laterdevelops into an adult gametophyte or gametophore that bears

    gametangia (d). Upon egg fertilization a diploid sporophyte is formed(e, marked by an arrowhead). Relatively abundant miRNAs, whichare differentially expressed between the stages a, c and e (Table 1)are indicated next to the stage with the highest expression (Axtell

    et al. 2007)

    Plant Mol Biol

    123

  • and Bowman (2004). They cloned the class III HD-ZIP

    gene homolog PpC3HDZIP1 from P. patens, and found

    that it contains a conserved miR166-target sequence.

    Using 50 rapid amplification of complementary DNA ends(50-RACE) they were able to clone a putative miR166-guided cleavage product of PpC3HDZIP1 mRNA (Floyd

    and Bowman 2004). This suggested that miR166 nega-

    tively regulates PpC3HDZIP1 expression in P. patens and

    provided indirect evidence for the presence of miR166 in

    this basal land plant.

    Soon after, the cloning of miR160 and miR160-guided

    cleavage products of auxin response factor (ARF) genes

    from the leafy gametophyte of the moss Polytrichum

    juniperinum was reported (Axtell and Bartel 2005). This

    provided direct evidence that miR160 is present and

    functional in moss. In A. thaliana, miR160 regulates

    ARF10, ARF16 and ARF17 (Liu et al. 2007; Mallory et al.

    2005; Wang et al. 2005) indicating that some miRNA:

    target interactions were deeply conserved during land plant

    evolution.

    Three groups used a conventional sRNA cloning

    approach to identify miRNAs from P. patens (Arazi et al.

    2005; Fattash et al. 2007; Talmor-Neiman et al. 2006a).

    Arazi et al. (2005) identified, among 100 cloned protonema

    (Fig. 1ac) sRNAs, homologs of miR390, miR156,

    miR319 and miR535, revealing their ancient origins within

    the land plant lineage. In addition, that study reported the

    cloning of five P. patens-specific miRNAs based on

    northern hybridization and the identification of corre-

    sponding pre-miRNAs among expressed sequence tags

    (ESTs) (Arazi et al. 2005). The release of P. patens whole-

    genome shotgun (WGS) sequences led to the identification

    of an additional 31 miRNA families, based on predicted

    hairpin structure surrounding cloned sRNAs or their

    homology to known miRNAs, from the same library

    (Talmor-Neiman et al. 2006a) and from a mixed protonema

    (Fig. 1a)-gametophore (Fig. 1cd) sRNA library (Fattash

    et al. 2007). These studies increased the number of iden-

    tified miRNA families in P. patens to 40, nine of which

    were deeply conserved with flowering plants.

    The introduction of next-generation sequencing tech-

    nology and the parallel completion of the P. patens draft

    genome sequence (Rensing et al. 2008) paved the way for a

    more comprehensive exploration of its miRNA repertoire.

    Deep sequencing of P. patens sRNAs from three major

    developmental stages (Fig. 1a, c, e) yielded 127,135

    unique sRNAs that matched the P. patens genome (Axtell

    et al. 2006). Computational analysis to predict pre-miRNA-

    like hairpin structures with suitable miRNA and miRNA*

    strands resulted in the confident identification of 97

    miRNA families (Axtell et al. 2007). These included three

    additional deeply conserved miRNAs and all except 11 of

    the previously identified miRNAs by Fattash et al. 2007.

    Two of those 11 miRNAs (Ppt-miR414 and Ppt-miR419)

    are currently under review and one (Ppt-miR167) has not

    yet been experimentally validated. Together, the above

    cloning efforts identified 233 P. patens miRNAs corre-

    sponding to 106 unique miRNA families. Of these, 13

    families (miR156, miR160, miR166, miR167, miR171,

    miR319, miR390, miR395, miR408, miR477, miR529,

    miR535, miR894) are conserved with one or more angio-

    sperms. Bryophytes are thought to have shared a common

    ancestor with flowering plants *400 MYA (Kenrick andCrane 1997) suggesting that these miRNAs have played

    regulatory roles since that period (Axtell and Bowman

    2008). One family (Ppt-miR536) is conserved with lyco-

    pods, and the remaining 93 families are considered moss-

    specific, since they have not yet been cloned from any

    other land plant. This number of miRNA families is

    comparable to the 131 miRNA families identified in

    A. thaliana (miRBase release version 16), indicating that

    the degree of miRNA regulation in moss, a basal plant, is at

    least equal to that in a flowering plant.

    As in flowering plants, P. patens miRNAs are encoded

    by either single (75/106) or multiple (31/106) genomic loci

    and their predicted pre-miRNAs vary is size and shape

    (Axtell et al. 2007; Fattash et al. 2007; Talmor-Neiman

    et al. 2006a). In addition, around 21% of them are arranged

    in closely linked clusters of two to three foldbacks that

    usually code for members of the same miRNA family

    (Axtell et al. 2007). This is comparable to the ratio found in

    a recent study of MIR gene clustering in A. thaliana, Pop-

    ulus and Oryza (Merchan et al. 2009). However, in contrast

    to angiosperm MIR genes that are usually found in inter-

    genic regions, around half of the P. patens pre-miRNAs

    overlap with the positions of protein-encoding loci as

    annotated in the draft genome assembly (Axtell et al. 2007).

    Axtell et al. (2007) noted that this arrangement is reminis-

    cent of MIR genes of the unicellular alga Chlamydomonas

    reinhardtii, many of which are found in introns of protein-

    encoding loci (Molnar et al. 2007; Zhao et al. 2007).

    Most cloned P. patens miRNAs (79%) are 21 nt long

    and start with a uracil (60%), similar to higher plant

    miRNAs. Nevertheless, a significant fraction of them

    (24%) start with a cytosine, compared to only 9% of

    A. thaliana miRNAs. This suggests a major role in the

    miRNA pathway for a P. patens AGO that favors 50 ter-minal cytosine and might represent a functional homolog

    of AtAGO5, previously shown to have a binding prefer-

    ence for sRNAs that initiate with cytosine (Mi et al. 2008).

    miRNA biogenesis in P. patens

    A. thaliana encodes four DICER-like (DCL) proteins

    (AtDCL1-4) that function with partial redundancy in

    Plant Mol Biol

    123

  • different sRNA pathways (Bouche et al. 2006; Deleris et al.

    2006; Gasciolli et al. 2005). AtDCL1 is the predominant

    DICER enzyme processing pre-miRNAs to release

    miRNA/miRNA* duplexes (Kurihara and Watanabe 2004;

    Park et al. 2002). Consistent with its primary role in

    miRNA maturation, Atdcl1 mutants are impaired in miRNA

    production (Kurihara et al. 2006; Kurihara and Watanabe

    2004; Park et al. 2002), ectopically express miRNA target

    genes (Kasschau et al. 2003) and show a range of devel-

    opmental phenotypes, including embryo lethality in com-

    plete loss-of-function mutants (Schauer et al. 2002). The

    P. patens genome encodes four DICER-like proteins

    (PpDCL), two of which (PpDCL1a and PpDCL1b) are

    related to AtDCL1, one to AtDCL3 and one to AtDCL4

    (Axtell et al. 2007). Khraiwesh et al. (2010) generated

    DPpDCL1a null mutants and found that they accumulatereduced miRNA and increased target mRNA levels, indi-

    cating that PpDCL1a is required for miRNA biogenesis and

    is a functional ortholog of AtDCL1. Furthermore,

    DPpDCL1a plants displayed severe developmental phe-notypes, including a protonema-stage arrest, emphasizing

    the importance of the miRNA pathway for normal game-

    tophyte development (Khraiwesh et al. 2010).

    The A. thaliana pre-miRNA hairpins are processed by

    AtDCL1 via at least two distinct mechanisms: a canonical

    loop-last mechanism (Kurihara et al. 2006; Kurihara and

    Watanabe 2004), and a loop-first mechanism currently

    found to be used for processing of the longer pre-miR159

    and pre-miR319 hairpins (Addo-Quaye et al. 2009; Bolo-

    gna et al. 2009). Degradome data suggest that both pro-

    cessing mechanisms exist in P. patens. Most P. patens

    hairpins are processed by the canonical loop-last mecha-

    nism (Addo-Quaye et al. 2009), whereas processing of the

    pre-miR319 precursors is conserved with A. thaliana and

    rice in being performed...

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